There are three related projects in this program: Project 1: Whole exome sequencing to identify DBA mutations. Project 2: In vitro culture and differentiation of DBA patient cells. Project 3: Small molecule screening and mechanistic studies of DBA. Project 1. The goal of this project is to provide a molecular diagnosis to each DBA patient. The first part of our genotyping screen is an analysis of ribosomal RNA (rRNA) processing in cultured peripheral blood lymphocytes. We have developed very sensitive metrics that can identify defects in both the large and small subunit ribosomal proteins (Farrar JE, Quarello P, Fisher R, O'Brien KA, Aspesi A, Parrella S, Henson AL, Seidel NE, Atsidaftos E, Prakash S, Bari S, Garelli E, Arceci RJ, Dianzani I, Ramenghi U, Vlachos A, Lipton JM, Bodine DM, Ellis SR. Exploiting pre-rRNA processing in Diamond Blackfan anemia gene discovery and diagnosis. Am J Hematol. 89(10):985-91, 2014). This can inform and confirm the targeted sequencing performed on all patients with a preliminary diagnosis of DBA. If no mutations are detected by targeted sequencing we proceed to SNParray analysis of deletions. We previously showed that deletions of ribosomal protein genes was responsible for about 10% of all DBA mutations (Farrar JE, Vlachos A, Atsidaftos E, Carlson-Donohoe H, Markello TC, Arceci RJ, Ellis SR, Lipton JM, Bodine DM. Ribosomal protein gene deletions in Diamond-Blackfan Anemia. Blood. 118 (26): 6943-51, 2011). These can include very small deletions that are well below the detection limit for cytogenetics (Vlachos A, Farrar JE, Atsidaftos E, Muir E, Narla A, Markello TC, Singh SA, Landowski M, Gazda HT, Blanc L, Liu JM, Ellis SR, Arceci RJ, Ebert BL, Bodine DM, Lipton JM. Diminutive somatic deletions in the 5q region lead to a phenotype atypical of classical 5q- syndrome. Blood. 122(14):2487-90, 2013). If no deletions are detected, we perform whole exome sequencing analysis of family groups consisting of parents, a DBA proband and either an affected or unaffected sibling. The inclusion of the parents and sibling added a great deal of power to our analysis allowing us to identify 2 strong candidate genes (MCM-2 and SEMA7A) in our first 8 families. We are currently validating these mutations in vitro and we are screening a pool of 96 undiagnosed DBA patients to see if they have any of the newly identified mutations. We have enrolled additional sets of families whose sequence is proceeding through the pipeline. However, our analysis of whole exome sequencing in the other 6 families we have completed has not identified an obvious candidate gene. We then hypothesized that the defect may lie outside the coding regions of the genome, and may involve deletions too small to be detected by SNP array. For families who have negative results with exome sequencing we have initiated 3 whole genome analyses designed to identify non-coding mutations, with an emphasis on deletions. Project 2: The goal of this project is to understand the specific lesions in DBA patients that prevent the generation of erythroid cells. We designed a two-step, 14 day in vitro culture system to generate erythroid cells from CD34+ stem/progenitor cells isolated from <10ml of peripheral blood (PB) collected from 12 patients enrolled in the DBAR. Patient cells were compared to healthy control PB CD34+ cells. At day 14, we routinely obtain at least 10x fewer CD235+ erythroid cells (proerythroblasts and basophilic erythroblasts) in DBA cultures vs. controls (1x106 vs. 1x107 from 1x104 CD34+ cells). Further, a 2-7 day delay in the acquisition of the erythroid cell surface marker CD235 was observed. Gene expression analyses was performed by Affymetrix GeneChip Human Gene ST Arrays and RNASeq to analyze protein coding and long non-coding RNA transcripts in cells from day 14 of erythroid cultures. Ingenuity pathway analysis (IPA) of the dysregulated genes between patients with ribosomal mutations, GATA1 mutations and controls revealed that many of the genes dysregulated in DBA were involved in multiple leukocyte migration and inflammatory signaling pathways, including the IL8, IL1R1, CXCR4, ICAM3, MPO, TNFSF10, and TLR4 genes with IL6, TNF, and lipopolysaccharide as top upstream regulators. Notably, the dysregulated genes in GATA1 patient cells largely overlapped that of the DBA patients with ribosomal mutations, including disruption of the leukocyte migration and inflammatory response genes. Patients with GATA1 and ribosomal protein mutations shared a number of dysregulated erythroid genes including AHSP, FAM132B, HEMGN, and TRIM10, however GATA1 patient cells showed GATA1 as the top upstream regulator and additionally showed dysregulation of heme biosynthesis pathway genes, including the ALAS2, FECH, CPOX, PPOX, and UROS. We are currently investigating the inflammatory pathways in DBA to identify novel targets for therapeutic development. Project 3. The goal of this project is to identify small molecules that can be used to treat DBA. The majority of DBA patients are heterozygous for a ribosomal protein gene carrying a deleterious mutation and a normal healthy gene. Haploinsufficeincy results because of decreased translation of functional RP protein. The RP mRNAs are among those RNAs with Terminal Oligo Pyrimidine (TOP) sequences in the 5UTR that regulates the rate of translation. We have developed a reporter cell line that has a luciferase gene with a TOP UTR. This cell line shows deceased translation of luciferase when treated with rapamycin, a known inhibitor of translation. In addition, serum starvation specifically inhibits the translation of TOP mRNAs. Our reporter cell line shows a 3-5-fold reduction in luciferase levels when serum starved. In collaboration with NCATS we are currently optimizing our reporter assay to fit a 1536 well plate format. Our plan is to treat serum starved cells with compounds and assay for increased luciferase output. We will screen small molecules to see which ones can increase the translation of TOP mRNA. These compounds will be further tested in patient cells and our mouse model (Devlin EE, Dacosta L, Mohandas N, Elliott G, Bodine DM. A transgenic mouse model demonstrates a dominant negative effect of a point mutation in the RPS19 gene associated with Diamond-Blackfan anemia. Blood. 116(15):2826-35, 2010.